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Old 01-09-2017, 08:55 AM   #1
schumric
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Location: Pullman, WA

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Default Question: bcftools: Wrong number of PL fields? nals=4 npl=6

Hello all,

I am new to bcftools and samtools, so forgive my question if it is ignorant. My goal is to extract consensus sequences between two different species.

I was given paired-end Illumina sequencing data. So far, I used FastQC to trim and bowtie 2 to perform the mapping and used samtools to generate sorted bam files. We created two bam files by using single-ended bowtie 2 mapping - this was due to a large amounts of insertion-deletion polymorphisms between the two species.

I tried the following to variant call:

/home/sean/Desktop/assembly_software/samtools-1.3.1/samtools mpileup -d8000 -uf Mlongifolia_CMEN585_assembly_v1.0.fasta CMEN585.R_1.sorted.bam CMEN585.R_2.sorted.bam | bcftools call -c | vcfutils.pl vcf2fq > cnsnew.fq

error: Wrong number of PL fields? nals=4 npl=6

Then I tried using just a single bam file:

/home/sean/Desktop/assembly_software/samtools-1.3.1/samtools mpileup -d8000 -uf Mlongifolia_CMEN585_assembly_v1.0.fasta CMEN585.R_1.sorted.bam | bcftools call -c | vcfutils.pl vcf2fq > cnsnew.fq

error: Wrong number of PL fields? nals=4 npl=3

What do these errors mean and how do I fix them? I have searched around, but have been unable to turn up anything so far.

I assume you need to variant call before you extract consensus sequences. I tried to extract the consensus sequences anyway (just for kicks):

/home/sean/Desktop/assembly_software/samtools-1.3.1/samtools \ mpileup \ -B \ -u \ -f Mlongifolia_CMEN585_assembly_v1.0.fasta\ CMEN585.R_1.sorted.bam | \ bcftools call -cg | \ vcfutils.pl vcf2fq \ ->R_1_consensus.fq

error: Use of uninitialized value $l in numeric lt (<) at /usr/local/bin/vcfutils.pl line 566. Use of uninitialized value $l in numeric lt (<) at /usr/local/bin/vcfutils.pl line 566.

Thank you for your help,

Richard
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Old 01-09-2017, 09:34 AM   #2
dpryan
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The bcftools error is usually due to mixing different versions of samtools and bcftools. If you use samtools 1.3.1, then you need bcftools 1.3.1.

I haven't a clue about the vcfutils error.
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Old 01-09-2017, 09:49 AM   #3
schumric
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Thank you for the reply. I tried to adjust the command so that it couldn't default to a different bcf tools version:

/home/sean/Desktop/assembly_software/samtools-1.3.1/samtools mpileup -d8000 -uf Mlongifolia_CMEN585_assembly_v1.0.fasta CMEN585.R_1.sorted.bam CMEN585.R_2.sorted.bam | /home/sean/Desktop/assembly_software/bcftools-1.3.1/bcftools call -c | vcfutils.pl vcf2fq > cnsnew.fq

Unfortunately, I got the same errors.

Note: Neither --ploidy nor --ploidy-file given, assuming all sites are diploid
[mpileup] 2 samples in 2 input files
Wrong number of PL fields? nals=4 npl=6

Richard
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Old 01-09-2017, 10:37 AM   #4
schumric
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I don't know if this helps, but I'm breaking down the steps currently to see what is failing.

1. /home/sean/Desktop/assembly_software/samtools-1.3.1/samtools mpileup -d8000 -uf Mlongifolia_CMEN585_assembly_v1.0.fasta CMEN585.R_1.sorted.bam CMEN585.R_2.sorted.bam >file.mpileup

2, /home/sean/Desktop/assembly_software/bcftools-1.3.1/bcftools call -c file.mpileup > file.vcf

3, /home/sean/Desktop/assembly_software/bcftools-1.3.1/vcfutils.pl vcf2fq file.vcf > cnsnew.fq

Currently, while running the first step I'm getting the message [mplp_func] Skipping because some_number is outside of 0 [ref:some_different_number. This might just be because the alignment is poor. I'm not sure.

Thank you
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